Data Sheet. PCSK9[Biotinylated]-LDLR Binding Assay Kit Catalog # 72002

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1 Data Sheet PCSK9[Biotinylated]-LDLR Binding Assay Kit Catalog # DESCRIPTION: The PCSK9[Biotinylated]-LDLR Binding Assay Kit is designed for screening and profiling purposes. PCSK9 is known to function as a negative regulator of hepatic lowdensity lipoprotein receptors (LDLRs) by binding to the LDLR ectodomain. The PCSK9[Biotinylated]-LDLR Binding Assay Kit comes in a convenient 96-well format, with biotinlabeled PCSK9, purified LDLR ectodomain, streptavidin labeled HRP, and assay buffer for 100 binding reactions. The key to this kit is the high sensitivity of detection of biotin-labeled PCSK9 by streptavidin-hrp. Only a few simple steps on a microtiter plate are required for the assay. First, LDLR ectodomain is coated on a 96-well plate. Next, PCSK9 is incubated with LDLR on the plate. Finally, the plate is treated with streptavidin-hrp followed by addition of an HRP substrate to produce chemiluminescence, which can then be measured using a chemiluminescence reader. COMPONENTS: Catalog # Component Amount Storage PCSK9, Biotinylated 10 µg -80 C LDLR 10 µg -80 C Streptavidin-HRP 15 µl +4 C 3x PCSK9 assay buffer 50 ml -20 C Blocking buffer 50 ml +4 C HRP chemiluminescent substrate 6 ml each +4 C (2 components) White 96-well microplate 1 +4 C (Avoid freeze/ thaw cycles!) MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED: PBS buffer Luminometer or fluorescent microplate reader capable of reading chemiluminescence Rotating or rocker platform APPLICATIONS: Great for screening small molecules and antibodies that inhibit the binding of PCSK9 to LDLR ectodomain.

2 STABILITY: One year from date of receipt when stored as directed. REFERENCES: 1. Chan, J.C. et al. (2009). Proc. Natl Acad. Sci. USA, 106, Liang, H., et al. (2012) J. Pharmacol. Exp. Ther ASSAY PROTOCOL: All samples and controls should be tested in duplicate. Coating the plate with LDLR: 1) Thaw LDLR on ice. Upon first thaw, briefly spin tube containing LDLR to recover the full contents of the tube. Aliquot into single use aliquots. Immediately store remaining LDLR in aliquots at -80 o C. Note: LDLR is very sensitive to freeze/thaw cycles. Avoid multiple freeze/thaw cycles. 2) Dilute LDLR to 2 µg/ml in PBS. 3) Add 50 µl of diluted LDLR solution to each well and incubate overnight at 4 o C. Leave a couple of wells empty (uncoated), for use with the Ligand Control (see below). 4) Dilute 3x PCSK9 Assay buffer to 1x PCSK9 Assay buffer with water. 5) Decant to remove supernatant. Wash the plate 3 times with 100 µl 1x PCSK9 Assay buffer. Tap plate onto clean paper towels to remove liquid. 6) Block wells by adding 100 µl of Blocking buffer to each well. Incubate for 1 hour at room temperature. Decant to remove supernatant. Step 1: 1) Prepare the master mixture: N wells x (10 µl 3x PCSK9 assay buffer + 15 µl H 2 O ) 2) Thaw PCSK9-biotin on ice. Upon first thaw, briefly spin tube containing enzyme to recover full contents of the tube. Aliquot PCSK9-biotin into single use aliquots. Immediately store remaining undiluted enzyme in aliquots at -80 C. Note: PCSK9 is very sensitive to freeze/thaw cycles. Do not re-use thawed aliquots or diluted enzyme. 3) Dilute PCSK9-biotin in 1x PCSK9 assay buffer at 2.5 ng/µl (50 ng/20 µl). Keep diluted protein on ice until use. Discard any unused diluted protein after use.

3 4) Add 25 µl of master mixture to each well. Use uncoated wells for the Ligand Control. Blank Ligand Control Positive Control Test Inhibitor 3x PCSK9 assay buffer 10 µl 10 µl 10 µl 10 µl H 2 O 15 µl 15 µl 15 µl 15 µl Test Inhibitor/Activator 5 µl Inhibitor buffer (no inhibitor) 5 µl 5 µl 5 µl 1x PCSK9 assay buffer 20 µl PCSK9-biotin (2.5 ng/µl) 20 µl 20 µl 20 µl Total 50 µl 50 µl 50 µl 50 µl 5) Add 5 µl of inhibitor solution to each well designated Test Inhibitor. For the Positive Control, Ligand Control and Blank, add 5 µl of the same solution without inhibitor (inhibitor buffer). 6) Add 20 µl of 1x PCSK9 Assay buffer to the well designated Blank. 7) Initiate reaction by adding 20 µl of diluted PCSK9 (see Step 1-3). Incubate at room temperature for two hours. 8) Decant to remove supernatant. Wash the plate 3 times with 100 µl 1x PCSK9 Assay buffer. Tap plate onto clean paper towels to remove liquid. 9) Block wells by adding 100 µl of Blocking buffer to each well. Incubate for 10 minutes at room temperature. Decant to remove supernatant. Step 2: 1) Dilute Streptavidin-HRP 1000-fold with Blocking buffer. 2) Add 100 µl to each well. Incubate for 1 hour at room temperature with slow shaking. 3) Wash plate three times with 1x PCSK9 assay buffer. Tap plate onto clean paper towels to remove liquid.

4 4) Block wells by adding 100 µl of Blocking Buffer to each well. Incubate for 10 minutes at room temperature. Decant to remove supernatant. Tap plate onto clean paper towels to remove liquid. 5) Just before use, mix on ice 50 µl HRP chemiluminescent substrate A and 50 µl HRP chemiluminescent substrate B, then add 100 µl to each well. Discard any unused chemiluminescent reagent after use. 6) Immediately read sample in a luminometer or microtiter-plate capable of reading chemiluminescence. Blank value is subtracted from all readings. Reading Chemiluminescence: Chemiluminescence is the emission of light (luminescence) which results from a chemical reaction. The detection of chemiluminescence requires no wavenlength selection because the method used is emission photometry and is not emission spectrophotometry. To properly read chemiluminescence, make sure the plate reader is set for LUMINESCENCE mode. Typical integration time is 1 second, delay after plate movement is 100 msec. Do not use a filter when measuring light emission. Typical settings for the Synergy 2 BioTek plate reader are: use the hole position on the filter wheel; Optics position: Top; Read type: endpoint. Sensitivity may be adjusted based on the luminescence of a control assay without enzyme (typically we set this value as 100). Example of Assay Results: Luminesence PCSK9[B]-LDLR Interaction ng PCSK9[B] LDLR Coated Wells No Coating luminesence Inhibition of PCSK9[B]-LDLR by Anti-PCSK IC50=0.4nM Log (Antibody) [nm]

5 PCSK9-LDLR binding activity, measured using the using the PCSK9[Biotinylated]-LDLR Binding Assay Kit, BPS Bioscience, Catalog #72002 (both) and Anti-PCSK9 Inhibitor (right). Luminescence was measured using a Bio-Tek fluorescent microplate reader. Data shown is lotspecific. For lot-specific information, please contact BPS Bioscience, Inc. at info@bpsbioscience.com RELATED PRODUCTS: Product Name Catalog # Size PCSK µg LDLR µg PCSK9-biotin µg LDLR-biotin µg PCSK9 Neutralizing Ab μg PCSK9-LDLR TR-FRET Assay Kit rxns. PCSK9(D374T)-LDLR TR-FRET Assay Kit rxns.

6 TROUBLESHOOTING GUIDE Problem Possible Cause Solution Luminescence signal of PCSK9 or LDLR has Enzyme loses activity upon repeated positive control reaction is weak lost activity freeze/thaw cycles. Use fresh PCSK9- biotin, (BPS Bioscience #71304) and fresh LDLR (BPS Bioscience #71205). Store proteins in single-use aliquots. Increase time of enzyme incubation. Increase enzyme concentration. Luminescent signal is erratic or varies widely among wells Background (signal to noise ratio) is high Antibody reaction is insufficient Incorrect settings on instruments Chemiluminescent reagents mixed too soon Inaccurate pipetting/technique Increase time for primary antibody incubation. Avoid freeze/thaw cycles of antibodies. Refer to instrument instructions for settings to increase sensitivity of light detection. Chemiluminescent solution should be used within 15 minutes of mixing. Ensure both reagents are properly mixed. Run duplicates of all reactions. Use a multichannel pipettor. Use master mixes to minimize errors. Bubbles in wells Pipette slowly to avoid bubble formation. Tap plate lightly to disperse bubbles; be careful not to splash between wells. Insufficient washes Increase number of washes. Increase wash volume. Increase Tween-20 concentration to 0.1% in TBST. Sample solvent is inhibiting the enzyme Results are outside the linear range of the assay Run negative control assay including solvent. Maintain DMSO level at <1% Increase time of enzyme incubation. Use different concentrations of PCSK9-biotin (BPS Bioscience #71304) to create a standard curve.

Data Sheet. PCSK9[Biotinylated]-LDLR Binding Assay Kit Catalog # 72002

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